Nucleic acid sequence encoding lymphoma associated molecule BAL

ABSTRACT

The invention provides isolated nucleic acids molecules, designated BAL nucleic acid molecules, which are differentially expressed in non-Hodgkin&#39;s lymphoma. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing BAL nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a BAL gene has been introduced or disrupted. The invention still further provides isolated BAL proteins, fusion proteins, antigenic peptides and anti-BAL antibodies. Diagnostic methods using compositions of the invention are also provided.

RELATED APPLICATIONS

This application is a national stage of PCT application no.PCT/US99/25439 filed on Oct. 29, 1999, which claims the benefit of U.S.Provisional application Ser. No. 60/106,383 filed on Oct. 29, 1998 andU.S. Provisional application Ser. No. 60/106,448 filed on Oct. 30, 1998.

GOVERNMENT FUNDING

Work described herein was supported, at least in part, under grant1P01CA66996-01A1 awarded by the NIH. The U.S. government therefore mayhave certain rights in this invention.

BACKGROUND OF THE INVENTION

The incidence of non-Hodgkin's lymphoma in the United States hasincreased by 75.1% between 1973 and 1992 (Kosary et al., SEER CancerStatistics Review, 1973-1992: Tables and Graphs, National CancerInstitute, NIH Publication No 96-2789, Bethesda, Md.: NIH 1995), apercentage increase exceeded only by that for prostate cancer, lungcancer in women, and melanoma.

Diffuse large B-cell lymphoma (DLB-CL) is the most common non-Hodgkin'slymphoma in adults. Although DLB-CL is curable in approximately 40% ofpatients, the majority of patients progress and die of their disease(Shipp et al. Non-Hodgkin's Lymphomas. In DeVita (ed): Principles andPractice of Oncology, 5th Edition, Philadelphia, J. B. LippincottCompany, pp. 2165-2220, 1997). Additional advancements in the treatmentof this aggressive but potentially curable non-Hodgkin's lymphoma arelikely to require a more precise understanding of the disease's cellularand molecular bases.

SUMMARY OF THE INVENTION

To identify genes which contribute to the observed differences inclinical outcome in DLB-CLs, the technique of differential display(Liang P. et al. (1992) Science, 257:967) was used in panels of primarytumors from patients with known clinical prognostic characteristics andmature follow-up. A novel 3′ cDNA, termed BAL, was found to besignificantly more abundant in tumors from patients with “high-risk(HR)” (International Prognostic Index, IPI) fatal disease than in tumorsfrom cured “low risk (LR [IPI])” patients (Shipp M. et al. (1993) N.Engl. J. Med., 329:987-994).

Accordingly, the present invention is based, at least in part, on thediscovery of novel molecules which are differentially expressed intumors from patients with “high risk” fatal DLB-CL disease or “low risk”cured DLB-CL. Their differentially expressed gene products are referredto herein as the “B-aggressive lymphoma” (“BAL”) nucleic acid andprotein. The BAL molecules of the present invention are useful asmodulating agents for regulating a variety of cellular processes.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding BAL proteins or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection of BAL-encoding nucleic acids.

In one embodiment, a BAL nucleic acid molecule of the invention is atleast 50%, 55%, 60%, 65%, 70%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, ormore identical to the nucleotide sequence (e.g., to the entire length ofthe nucleotide sequence) shown in SEQ ID NO:1, 3, 4, or 6.

In a preferred embodiment, the isolated nucleic acid molecule includesthe nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof.In another embodiment, the nucleic acid molecule includes SEQ ID NO:3and nucleotides 1-228 of SEQ ID NO:1. In another embodiment, the nucleicacid molecule includes SEQ ID NO:3 and nucleotides 2791-3243 of SEQ IDNO:1. In another preferred embodiment, the nucleic acid moleculeconsists of the nucleotide sequence shown in SEQ ID NO:1 or 3. Inanother preferred embodiment, the nucleic acid molecule includes afragment of at least 607 nucleotides (e.g., 607 contiguous nucleotides)of the nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof.

In another preferred embodiment, the isolated nucleic acid moleculeincludes the nucleotide sequence shown SEQ ID NO:4 or 6, or a complementthereof. In another embodiment, the nucleic acid molecule includes SEQID NO:6 and nucleotides 1-170 of SEQ ID NO:4. In another embodiment, thenucleic acid molecule includes SEQ ID NO:6 and nucleotides 2649-3024 ofSEQ ID NO:4. In another preferred embodiment the nucleic acid moleculeconsists of the nucleotide sequence shown in SEQ ID NO:4 or 6. Inanother preferred embodiment, the nucleic acid molecule includes afragment of at least 452 nucleotides (e.g., 452 contiguous nucleotides)of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, or a complementthereof.

In another embodiment, a BAL nucleic acid molecule includes a nucleotidesequence encoding a protein having an amino acid sequence sufficientlyhomologous to the amino acid sequence of SEQ ID NO:2 or 5. In apreferred embodiment, a BAL nucleic acid molecule includes a nucleotidesequence encoding a protein having an amino acid sequence at least 50%,55%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more homologousto the entire length of the amino acid sequence of SEQ ID NO:2 or 5.

In another preferred embodiment, an isolated nucleic acid moleculeencodes the amino acid sequence of human BAL. In yet another preferredembodiment, the nucleic acid molecule includes a nucleotide sequenceencoding a protein having the amino acid sequence of SEQ ID NO:2 or 5.In yet another preferred embodiment, the nucleic acid molecule is atleast 452 or 607 nucleotides in length. In a further preferredembodiment, the nucleic acid molecule is at least 452 or 607 nucleotidesin length and encodes a protein having a BAL activity (as describedherein).

Another embodiment of the invention features nucleic acid molecules,preferably BAL nucleic acid molecules, which specifically detect BALnucleic acid molecules relative to nucleic acid molecules encodingnon-BAL proteins. For example, in one embodiment, such a nucleic acidmolecule is at least 300-350, 350-400, 400-450, 452, 452-500, 500-550,550-600, 607 or more nucleotides in length and hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence shown in SEQ ID NO:1 or 4 or a complement thereof.

In preferred embodiments, the nucleic acid molecules are at least 15(e.g., contiguous) nucleotides in length and hybridize under stringentconditions to nucleotides 1-333, 351-385, 463-824, 931-1082, or3232-3244 of SEQ ID NO:1. In other preferred embodiments, the nucleicacid molecules comprise nucleotides 1-333, 351-385, 463-824, 931-1082,or 3232-3244 of SEQ ID NO:1.

In other preferred embodiments, the nucleic acid molecules are at least15 (e.g., contiguous) nucleotides in length and hybridize understringent conditions to nucleotides 1-39 or 57-1841 of SEQ ID NO:4. Inother preferred embodiments, the nucleic acid molecules comprisenucleotides 1-39 or 57-1841 of SEQ ID NO:4.

In other preferred embodiments, the nucleic acid molecule encodes anaturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:2 or 5, wherein the nucleic acidmolecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1,3, 4, or 6 under stringent conditions.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a BAL nucleic acid molecule, e.g., thecoding strand of a BAL nucleic acid molecule.

Another aspect of the invention provides a vector comprising a BALnucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. In yetanother embodiment, the invention provides a host cell containing anucleic acid molecule of the invention. The invention also provides amethod for producing a protein, preferably a BAL protein, by culturingin a suitable medium, a host cell, e.g., a mammalian host cell such as anon-human mammalian cell, of the invention containing a recombinantexpression vector, such that the protein is produced.

Another aspect of this invention features isolated or recombinant BALproteins and polypeptides. In one embodiment, the isolated protein,preferably a BAL protein, includes at least one proline rich domain. Ina preferred embodiment, the isolated protein, preferably a BAL protein,includes at least one proline rich domain and at least one tyrosinephosphorylation site. In a preferred embodiment, the protein, preferablya BAL protein, includes at least one proline rich domain, at least onetyrosine phosphorylation site, and has an amino acid sequence at leastabout 50%, 55%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or morehomologous to the amino acid sequence of SEQ ID NO:2 or 5. In anotherpreferred embodiment, the protein, preferably a BAL protein, includes atleast one proline rich domain, at least one tyrosine phosphorylationsite, and plays a role in the pathogenesis of non-Hodgkin's lymphoma. Inyet another preferred embodiment, the protein, preferably a BAL protein,includes at least one proline rich domain, at least one tyrosinephosphorylation site, and is encoded by a nucleic acid molecule having anucleotide sequence which hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1, 3, 4, or 6.

In another embodiment, the invention features BAL proteins which areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a BAL protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques, based on the amino acidsequence of SEQ ID NO:2 or 5.

In another embodiment, the invention features fragments of the proteinhaving the amino acid sequence of SEQ ID NO:2 or 5, wherein the fragmentcomprises at least 15 amino acids (e.g., contiguous amino acids) of theamino acid sequence of SEQ ID NO:2 or 5. In another embodiment, theprotein, preferably a BAL protein, has the amino acid sequence of SEQ IDNO:2 or 5, respectively.

In another embodiment, the invention features an isolated protein,preferably a BAL protein, which is encoded by a nucleic acid moleculeconsisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%,70%, 72%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to anucleotide sequence of SEQ ID NO:1, 3, 4, or 6, or a complement thereof.This invention further features an isolated protein, preferably a BALprotein, which is encoded by a nucleic acid molecule consisting of anucleotide sequence which hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1, 3, 4, or 6, or a complement thereof.

The proteins of the present invention or portions thereof, e.g.,biologically active portions thereof, can be operatively linked to anon-BAL polypeptide (e g., heterologous amino acid sequences) to formfusion proteins. The invention further features antibodies, such asmonoclonal or polyclonal antibodies, that specifically bind proteins ofthe invention, preferably BAL proteins. In addition, the BAL proteins orbiologically active portions thereof can be incorporated intopharmaceutical compositions, which optionally include pharmaceuticallyacceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of a BAL nucleic acid molecule, protein or polypeptide in abiological sample by contacting the biological sample with an agentcapable of detecting a BAL nucleic acid molecule, protein or polypeptidesuch that the presence of a BAL nucleic acid molecule, protein orpolypeptide is detected in the biological sample.

In another aspect, the present invention provides a method for detectingthe presence of BAL activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator of BALactivity such that the presence of BAL activity is detected in thebiological sample.

In another aspect, the invention provides a method for modulating BALactivity comprising contacting a cell capable of expressing BAL with anagent that modulates BAL activity such that BAL activity in the cell ismodulated. In one embodiment, the agent inhibits BAL activity. Inanother embodiment, the agent stimulates BAL activity. In oneembodiment, the agent is an antibody that specifically binds to a BALprotein. In another embodiment, the agent modulates expression of BAL bymodulating transcription of a BAL gene or translation of a BAL mRNA. Inyet another embodiment, the agent is a nucleic acid molecule having anucleotide sequence that is antisense to the coding strand of a BAL mRNAor a BAL gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant BAL proteinor nucleic acid expression or activity, e.g., non-Hodgkin's lymphoma, byadministering an agent which is a BAL modulator to the subject. In oneembodiment, the BAL modulator is a BAL protein. In another embodimentthe BAL modulator is a BAL nucleic acid molecule. In yet anotherembodiment, the BAL modulator is a peptide, peptidomimetic, or othersmall molecule. In a preferred embodiment, the disorder characterized byaberrant BAL protein or nucleic acid expression is a proliferativedisorder, e.g., non-Hodgkin's lymphoma.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aBAL protein; (ii) mis-regulation of the gene; and (iii) aberrantpost-translational modification of a BAL protein, wherein a wild-typeform of the gene encodes a protein with a BAL activity.

In another aspect the invention provides a method for producing oridentifying a compound that binds to or modulates the activity of a BALprotein, by providing an indicator composition comprising a BAL proteinhaving BAL activity, contacting the indicator composition with a testcompound, and determining the effect of the test compound on BALactivity in the indicator composition to produce or identify a compoundthat modulates the activity of a BAL protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cDNA sequence and predicted amino acid sequence ofhuman BAL. The nucleotide sequence corresponds to nucleic acids 1 to3243 of SEQ ID NO:1. The amino acid sequence corresponds to amino acids1 to 826 of SEQ ID NO: 2. The coding region without the 5′ and 3′untranslated regions of the human BAL gene is shown in SEQ ID NO:3.

FIG. 2 depicts the cDNA sequence and predicted amino acid sequence ofmurine BAL. The nucleotide sequence corresponds to nucleic acids 1 to3024 of SEQ ID NO:4. The amino acid sequence corresponds to amino acids1 to 826 of SEQ ID NO: 5. The coding region without the 5′ and 3′untranslated regions of the murine BAL gene is shown in SEQ ID NO:6.

FIG. 3 depicts an alignment of the human BAL protein with the murine BALprotein using the ALIGN program (version 2.0), a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 2.

FIG. 4 depicts an alignment of the human BAL nucleic acid molecule withthe murine BAL nucleic acid molecule using the ALIGN program (version2.0), a PAM120 weight residue table, a gap length penalty of 16 and agap penalty of 4.

FIG. 5 depicts a northern blot analysis of total RNA from 5 DLB-CL celllines, 4 “high-risk” and 5 “low-risk” primary tumors, and 2 pairs ofnormal B-splenocytes, with or without Ig-activation. A band of ˜3.2 kb(arrow), corresponding to the BAL message, is detected at high levels inonly one of the cell lines (DHL-7), in the “high-risk” tumors andIg-activated splenocytes. BAL transcripts are less abundant in the“low-risk” primary tumors. Likewise, BAL is expressed at lower levels inthe non-activated B-cells. The β-actin blot demonstrates that loadingdoes not account for the differences in BAL expression.

FIG. 6 depicts a northern blot comparing BAL transcripts in a tumorderived from a DLB-CL cell line grown in SCID mouse and the parentalsuspension cells. BAL is expressed at significantly higher levels in thetumor derived RNA than in the parental cell line. The β-actin blotdemonstrates that loading does not account for the observed differences.

FIG. 7 depicts a diagrammatic representation of BAL cDNA and proteinstructure. Two alternatively spliced BAL cDNAs of 3243 bp (BALL, long)and 3138 bp (BAL_(S), short) encode previously uncharacterized 854 aaand 819 aa proteins. The alternatively spliced region and the start andstop codons are indicated in BAL cDNA. The region of partial homology tothe non-histone region of histone macroH2A and the potential CRK/CRK-1binding sites (YHVL, YSVP) and proline-rich region are also highlighted.

FIG. 8 depicts a northern blot analysis of BAL transcripts in multiplehuman tissues. BAL transcripts are most abundant in analyzedlymphoid/hematopoietic tissues (spleen, lymph node, fetal liver andperipheral blood) and several non-hematopoietic organs (heart, skeletalmuscle and colon).

FIG. 9 depicts FISH analysis of a normal human metaphase with genomicPAC clones encompassing the BAL locus. The top panel shows theGiemsa-banded metaphase. In the FISH panel (lower), the green signalsrepresent the BAL-PAC clones hybridized to the two normal chromosome3q21.

FIG. 10 depicts sensitivity of the BAL semi-quantitative duplex RT-PCR.The abundance of BAL in a given sample was determined by comparing theintensity of co-amplified BAL and control (ABL) PCR products by scanningdensitometry. The sensitivity of the duplex PCR was determined by mixingfixed amounts of cDNAs from a BAL negative and a BAL positive DLB-CLcell line to mimic BAL losses of 10%-100% (lower panel). When the ratioof BAL/ABL signals was plotted against the percentage of BAL lost in agiven sample, the data yield a straight line and a r² value of 0.967(top panel).

FIG. 11 depicts the results from the duplex PCR experiments.

FIG. 12 is a graph depicting BAL expression as determined by the duplexPCR experiments (quantified with scanning densitometry).

FIG. 13 depicts the results from a western blot analysis of aggressivelymphoma transfectants expressing pEGFP vector only or pEGFP-BAL_(S).

FIG. 14 is a graph depicting the cell migration of pEGFP vector only orpEGFP-BAL_(S) transfectants.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as BAL nucleic acid and proteinmolecules which are differentially expressed in malignancies such aslymphoma, e.g., non-Hodgkin's lymphoma. The newly identified BAL nucleicacid and protein molecules can be used to identify cells exhibiting orpredisposed to a malignancy such as lymphoma, e.g., non-Hodgkin'slymphoma, thereby diagnosing subjects having, or prone to developingsuch disorders.

As used herein, a “malignancy” includes a cancerous uncontrolled growthof cells in an area of the body. Malignant cancers are typicallyclassified by their microscopic appearance and the type of tissue fromwhich they arise. Examples of malignancies include carcinomas, sarcomas,myelomas, chondrosarcomas, adenosarcomas, angiosarcomas, neuroblastomas,gliomas, medulloblastomas, erythroleukemias, and myelogenous leukemias.

As used herein, a “lymphoma” includes a malignant neoplastic disorder oflymphoreticular tissue which produces a distinct tumor mass. Lymphomasinclude tumors derived from the lymphoid lineage. Lymphomas usuallyarise in lymph nodes, the spleen, or other areas rich in lymphoidtissue. Lymphomas are typically subclassified as Hodgkin's disease andNon-Hodgkin's lymphomas, e.g., Burkitt's lymphoma, large-cell lymphoma,and follicular lymphoma.

As used herein, “differential expression” or differentially expressed”includes both quantitative as well as qualitative differences in thetemporal and/or cellular expression pattern of a gene, e.g., the BALgene, among, for example, normal cells and cells from patients with“high risk” fatal DLB-CL disease or “low risk” cured DLB-CL. Genes whichare differentially expressed can be used as part of a prognostic ordiagnostic marker for the evaluation of subjects at risk for developinga malignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma. Dependingon the expression level of the gene, the progression state or theaggressiveness of the disorder can be evaluated. Methods for detectingthe differential expression of a gene are described herein.

The BAL molecules comprise a family of molecules having certainconserved structural and functional features. The term “family” whenreferring to the protein and nucleic acid molecules of the invention isintended to mean two or more proteins or nucleic acid molecules having acommon structural domain or motif and having sufficient amino acid ornucleotide sequence homology as defined herein. Such family members canbe naturally or non-naturally occurring and can be from either the sameor different species. For example, a family can contain a first proteinof human origin, as well as other, distinct proteins of human origin oralternatively, can contain homologues of non-human origin. Members of afamily may also have common functional characteristics.

For example, the family of BAL proteins comprise at least one “prolinerich domain.” As used herein, the term “proline rich domain” includes anamino acid sequence of about 4-6 amino acid residues in length havingthe general sequence X-Pro-X-X-Pro-X (where X can be any amino acid).Proline rich domains are usually located in a helical structure and bindthrough hydrophobic interactions to SH3 domains. SH3 domains recognizeproline rich domains in both forward and reverse orientations. Prolinerich domains are described in, for example, Sattler M. et al., Leukemia(1998) 12:637-644, the contents of which are incorporated herein byreference. BAL proteins of the invention preferably include at least oneproline rich domain, but may contain two or more. Amino acid residues781-786 of the human BAL and amino acid residues 748-753 of the murineBAL comprise proline rich domains.

In another embodiment, a BAL protein of the present invention isidentified based on the presence of at least one “tyrosinephosphorylation site” in the protein or corresponding nucleic acidmolecule. As used herein, the term “tyrosine phosphorylation site”includes an amino acid sequence of about 4 amino acid residues in lengthhaving the general sequence Tyr-X-X-X (where X can be any amino acid).The tyrosine in this domain is phosphorylated in response to a cellularstimulus, for example, in response to a hematopoietic growth factor(e.g., thrombopoietin, erythropoietin, or steel factor) stimulation.Tyrosine phosphorylation of cellular proteins plays a major role in cellsignaling, e.g., hematopoietic cell signaling. Tyrosine phosphorylationsites are described in, for example, Sattler M. et al., Leukemia (1998)12:637-644, the contents of which are incorporated herein by reference.BAL proteins of the invention include at least one or two tyrosinephosphorylation sites, but may contain three or more. Amino acidresidues 392-395 and 495-498 of the human BAL comprise tyrosinephosphorylation sites.

In another embodiment, a BAL protein of the present invention isidentified based on the presence of at least one “rod domain” in theprotein or corresponding nucleic acid molecule. As used herein, the term“rod domain” includes an α-helical, filament forming structure that ismade up of smaller repeating structures. The smallest repeatingstructure may contain seven amino acids in which small, generallyhydrophobic amino acids are typically found in the first and fourthpositions of the repeat. The seven amino acids form two turns of anα-helix and the first and fourth positions fall in the hydrophobicinterior of the α-helix. Rod domains in alpha and beta cardiac myosinare described in, for example, Warrick et al., (1987) Annual Rev. CellBiol. 3:379-421.

In another embodiment, a BAL protein of the present invention isidentified based on the presence of at least one “α-helical region” inthe protein or corresponding nucleic acid molecule that is homologous tothe α-helical region of moesin, the most abundant ezrin-radixin-moesin(ERM) protein in lymphocytes. As used herein, the term “α-helicalregion” includes an amino acid sequence of about 10 to about 20 aminoacids in length that forms an α-helix. The α-helical region ofezrin-radixin-moesin (ERM) proteins is described in, for example,Bretscher (1999) Current Biology 8(12):721-4.

Due to their homology to protein families such as myosin heavy chain andcytoskeleton linkers ezrin-radixin-moesin, the human BAL molecules mayalso be involved in cellular functions such as cell migration, motility,and shape, as well as in cell/cell and cell/extra-cellular matrixinteractions through adhesion molecules.

Isolated proteins of the present invention, preferably BAL proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:2 or 5 or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:1, 3, 4, or 6. As used herein, theterm “sufficiently homologous” refers to a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains or motifs and/or acommon functional activity. For example, amino acid or nucleotidesequences which share common structural domains have at least 30%, 40%,or 50% homology, preferably 60% homology, more preferably 70%-80%, andeven more preferably 90-95% homology across the amino acid sequences ofthe domains and contain at least one and preferably two structuraldomains or motifs, are defined herein as sufficiently homologous.Furthermore, amino acid or nucleotide sequences which share at least30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95%homology and share a common functional activity are defined herein assufficiently homologous.

As used interchangeably herein, “BAL activity”, “biological activity ofBAL” or “functional activity of BAL”, refers to an activity exerted by aBAL protein, polypeptide or nucleic acid molecule on a BAL responsivecell or on a BAL protein substrate, as determined in vivo, ex vivo, orin vitro, according to standard techniques. In one embodiment, a BALactivity is a direct activity, such as an association with a BAL-targetmolecule. As used herein, a “target molecule” or “binding partner” is amolecule with which a BAL protein binds or interacts in nature, suchthat BAL-mediated function is achieved. A BAL target molecule can be anon-BAL molecule or a BAL protein or polypeptide of the presentinvention. In an exemplary embodiment, a BAL target molecule is a BALligand. Alternatively, a BAL activity is an indirect activity, such as acellular signaling activity mediated by interaction of the BAL proteinwith a BAL ligand. BAL activities include modulation of cellularadhesion and modulation of the aggressiveness or severity of amalignancy such as DLB-CL. BAL activities are described herein.

Accordingly, another embodiment of the invention features isolated BALproteins and polypeptides having a BAL activity. Preferred proteins areBAL proteins having at least one tyrosine phosphorylation site and,preferably, a BAL activity. Other preferred proteins are BAL proteinshaving at least one proline rich domain and, preferably, a BAL activity.Other preferred proteins are BAL proteins having at least one tyrosinephosphorylation site and/or at least one proline rich domain, and are,preferably, encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,3, 4, or 6.

The nucleotide sequence of the isolated human BAL cDNA and the predictedamino acid sequence of the human BAL polypeptide are shown in FIG. 1 andin SEQ ID NOs:1 and 2, respectively.

The human BAL gene, which is approximately 3244 nucleotides in length,encodes a protein having a molecular weight of approximately 95 kD andwhich is approximately 826 amino acid residues in length. On a multipletissue northern blot, BAL transcripts were most abundant in lymphoidorgans (spleen, lymph node, fetal liver, and peripheral blood) andseveral additional non-hematopoietic organs (heart, skeletal muscle andcolon).

The nucleotide sequence of the isolated murine BAL cDNA and thepredicted amino acid sequence of the murine polypeptide are shown inFIG. 2 and in SEQ ID NOs:4 and 5, respectively.

The murine BAL gene, which is approximately 3024 nucleotides in length,encodes a protein having a molecular weight of approximately 95 kD andwhich is approximately 826 amino acid residues in length.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode BAL proteins or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify BAL-encoding nucleic acid molecules (e.g., BAL mRNA)and fragments for use as PCR primers for the amplification or mutationof BAL nucleic acid molecules. As used herein, the term “nucleic acidmolecule” is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated BAL nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 3, 4, or 6 or aportion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all orportion of the nucleic acid sequence of SEQ ID NO:1, 3, 4, or 6, as ahybridization probe, BAL nucleic acid molecules can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook, J., Fritsh, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, 3, 4, or 6 can be isolated by the polymerase chain reaction(PCR) using synthetic oligonucleotide primers designed based upon thesequence of SEQ ID NO:1, 3, 4, or 6.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to BAL nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1. Thesequence of SEQ ID NO:1 corresponds to the human BAL cDNA. This cDNAcomprises sequences encoding the human BAL protein (i.e., “the codingregion”, from nucleotides 229-2790), as well as 5′ untranslatedsequences (nucleotides 1-228) and 3′ untranslated sequences (nucleotides2791-3243). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:1 (e.g., nucleotides 229-2790,corresponding to SEQ ID NO:3).

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:4. Thesequence of SEQ ID NO:4 corresponds to the murine BAL cDNA. This cDNAcomprises sequences encoding the murine BAL protein (i.e., “the codingregion”, from nucleotides 171-2648), as well as 5′ untranslatedsequences (nucleotides 1-170) and 3′ untranslated sequences (nucleotides2649-3024). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:4 (e.g., nucleotides 171-2648,corresponding to SEQ ID NO:6).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6, or a portionof any of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1, 3, 4, or6, is one which is sufficiently complementary to the nucleotide sequenceshown in SEQ ID NO:1, 3, 4, or 6, such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 72%, 75%, 80%, 85%, 90%, 95%, 98%or more homologous to the entire length of the nucleotide sequence shownin SEQ ID NO:1, 3, 4, or 6 or a portion of any of these nucleotidesequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, 3, 4, or 6, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of a BAL protein, e.g., a biologically active portionof a BAL protein. The nucleotide sequence determined from the cloning ofthe BAL gene allows for the generation of probes and primers designedfor use in identifying and/or cloning other BAL family members, as wellas BAL homologues from other species. The probe/primer typicallycomprises substantially purified oligonucleotide. The oligonucleotidetypically comprises a region of nucleotide sequence that hybridizesunder stringent conditions to at least about 12 or 15, preferably about20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense sequence of SEQ ID NO:1, 3, 4, or 6,of an anti-sense sequence of SEQ ID NO:1, 3, 4, or 6, or of a naturallyoccurring allelic variant or mutant of SEQ ID NO:1, 3, 4, or 6. In anexemplary embodiment, a nucleic acid molecule of the present inventioncomprises a nucleotide sequence which is greater than 300-350, 350-400,400-450, 452, 452-500, 500-550, 550-600, 607, 607-650, 650-700, 700-750,750-800, 800-850, 850-900, 900-950, 950-1000, or more nucleotides inlength and hybridizes under stringent hybridization conditions to anucleic acid molecule of SEQ ID NO:1, 3, 4, or 6.

Probes based on the BAL nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a BAL protein, such as by measuring a level of aBAL-encoding nucleic acid in a sample of cells from a subject e.g.,detecting BAL mRNA levels or determining whether a genomic BAL gene hasbeen mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of a BALprotein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:1, 3, 4, or 6, which encodes a polypeptide havinga BAL biological activity (the biological activities of the BAL proteinsare described herein), expressing the encoded portion of the BAL protein(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of the BAL protein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6, due todegeneracy of the genetic code and thus encode the same BAL proteins asthose encoded by the nucleotide sequence shown in SEQ ID NO:1, 3, 4, or6. In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence shown in SEQ ID NO:2 or 5.

In addition to the BAL nucleotide sequences shown in SEQ ID NO:1, 3, 4,or 6, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof the BAL proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the BAL genes may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a BALprotein, preferably a mammalian BAL protein, and can further includenon-coding regulatory sequences, and introns.

Allelic variants of human BAL include both fictional and non-functionalBAL proteins. Functional allelic variants are naturally occurring aminoacid sequence variants of the human BAL protein that maintain theability to bind a BAL ligand and/or modulate the occurrence or severityof a malignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma.Functional allelic variants will typically contain only conservativesubstitution of one or more amino acids of SEQ ID NO:2 or 5 orsubstitution, deletion or insertion of non-critical residues innon-critical regions of the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human BAL protein that do not have the abilityto either bind a BAL ligand and/or modulate occurrence or severity of amalignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma.Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion or prematuretruncation of the amino acid sequence of SEQ ID NO:2 or 5 or asubstitution, insertion or deletion in critical residues or criticalregions.

The present invention further provides non-human orthologues of thehuman BAL protein. Orthologues of the human BAL protein are proteinsthat are isolated from non-human organisms and possess the same BALligand binding and/or modulation of the occurrence or severity of amalignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma capabilitiesof the human BAL protein. Orthologues of the human BAL protein canreadily be identified as comprising an amino acid sequence that issubstantially homologous to SEQ ID NO:2 or 5.

Moreover, nucleic acid molecules encoding other BAL family members and,thus, which have a nucleotide sequence which differs from the BALsequences of SEQ ID NO:1, 3, 4, or 6 are intended to be within the scopeof the invention. For example, another BAL cDNA can be identified basedon the nucleotide sequence of human BAL. Moreover, nucleic acidmolecules encoding BAL proteins from different species, and which, thus,have a nucleotide sequence which differs from the BAL sequences of SEQID NO:1, 3, 4, or 6 are intended to be within the scope of theinvention. For example, a monkey BAL cDNA can be identified based on thenucleotide sequence of a human or murine BAL.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the BAL cDNAs of the invention can be isolated based ontheir homology to the BAL nucleic acids disclosed herein using the cDNAsdisclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the BAL cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the BAL gene.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, 3, 4, or 6. In otherembodiment, the nucleic acid is at least 30, 50, 100, 150, 200, 250,300, 350, 400, 450, 452, 500, 550, 607, 600, 650, 700, 750, 800, 850,900, or 950 nucleotides in length. As used herein, the term “hybridizesunder stringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%homologous to each other typically remain hybridized to each other.Preferably, the conditions are such that sequences at least about 70%,more preferably at least about 80%, even more preferably at least about85% or 90% homologous to each other typically remain hybridized to eachother. Such stringent conditions are known to those skilled in the artand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., morepreferably at 60° C., and even more preferably at 65° C. Preferably, anisolated nucleic acid molecule of the invention that hybridizes understringent conditions to the sequence of SEQ ID NO:1,3, 4, or 6corresponds to a naturally-occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the BAL sequencesthat may exist in the population, the skilled artisan will furtherappreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:1, 3, 4, or 6 thereby leading tochanges in the amino acid sequence of the encoded BAL proteins, withoutaltering the functional ability of the BAL proteins. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1, 3, 4, or 6. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of BAL (e.g., thesequence of SEQ ID NO:2 or 5) without altering the biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. For example, amino acid residues that are conserved among theBAL proteins of the present invention, are predicted to be particularlyunamenable to alteration. Furthermore, additional amino acid residuesthat are conserved between the BAL proteins of the present invention andother members of the BAL family are not likely to be amenable toalteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding BAL proteins that contain changes in amino acidresidues that are not essential for activity. Such BAL proteins differin amino acid sequence from SEQ ID NO:2 or 5, yet retain biologicalactivity. In one embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least about 50%, 55%, 60%, 62%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:2 or5.

An isolated nucleic acid molecule encoding a BAL protein homologous tothe protein of SEQ ID NO:2 or 5 can be created by introducing one ormore nucleotide substitutions, additions or deletions into thenucleotide sequence of SEQ ID NO:1, 3, 4, or 6, such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations can be introduced into SEQ ID NO:1, 3, 4, or6, by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a BALprotein is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a BAL coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for BAL biological activity to identify mutants that retainactivity. Following mutagenesis of SEQ ID NO:1, 3, 4, or 6, the encodedprotein can be expressed recombinantly and the activity of the proteincan be determined.

In a preferred embodiment, a mutant BAL protein can be assayed for theability to (1) interact with a non-BAL protein molecule, e.g., CRK,CRK-L, SHP-2, PBK, or ZAP70; (2) activate a BAL-dependent signaltransduction pathway; or (3) modulate the occurrence or severity of alymphoma, e.g., non Hodgkin's lymphoma.

In addition to the nucleic acid molecules encoding BAL proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire BAL coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding BAL. Theterm “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the coding region of human and murine BAL corresponds to SEQ ID NO:3 and6, respectively). In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding BAL. The term “noncoding region” refers to5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences encoding BAL disclosed herein (e.g.,SEQ ID NO:3 and 6), antisense nucleic acids of the invention can bedesigned according to the rules of Watson and Crick base pairing. Theantisense nucleic acid molecule can be complementary to the entirecoding region of BAL mRNA, but more preferably is an oligonucleotidewhich is antisense to only a portion of the coding or noncoding regionof BAL mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofBAL mRNA. An antisense oligonucleotide can be, for example, about 5, 10,15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a BAL proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveBAL mRNA transcripts to thereby inhibit translation of BAL mRNA. Aribozyme having specificity for a BAL-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a BAL cDNA disclosedherein (i e., SEQ ID NO:1, 3, 4, or 6). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a BAL-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,BAL mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, BAL gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the BAL(e.g., the BAL promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the BAL gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

In yet another embodiment, the BAL nucleic acid molecules of the presentinvention can be modified at the base moiety, sugar moiety or phosphatebackbone to improve, e.g., the stability, hybridization, or solubilityof the molecule. For example, the deoxyribose phosphate backbone of thenucleic acid molecules can be modified to generate peptide nucleic acids(see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1):5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” referto nucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc.Natl. Acad. Sci. 93: 14670-675.

PNAs of BAL nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of BAL nucleic acid molecules can also be used in theanalysis of single base pair mutations in a gene, (e.g., by PNA-directedPCR clamping); as ‘artificial restriction enzymes’ when used incombination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996)supra)); or as probes or primers for DNA sequencing or hybridization(Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In another embodiment, PNAs of BAL can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of BAL nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup B. (1996) supra and Finn P. J. et al.(1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain canbe synthesized on a solid support using standard phosphoramiditecoupling chemistry and modified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

II. Isolated BAL Proteins and Anti-BAL Antibodies

One aspect of the invention pertains to isolated BAL proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-BAL antibodies. In oneembodiment, native BAL proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, BAL proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a BAL protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the BALprotein is derived, or substantially free from chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of BAL protein in whichthe protein is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof BAL protein having less than about 30% (by dry weight) of non-BALprotein (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-BAL protein, still more preferablyless than about 10% of non-BAL protein, and most preferably less thanabout 5% non-BAL protein. When the BAL protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of BAL protein in which the protein isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of BAL protein having less than about 30% (by dry weight)of chemical precursors or non-BAL chemicals, more preferably less thanabout 20% chemical precursors or non-BAL chemicals, still morepreferably less than about 10% chemical precursors or non-BAL chemicals,and most preferably less than about 5% chemical precursors or non-BALchemicals.

As used herein, a “biologically active portion” of a BAL proteinincludes a fragment of a BAL protein which participates in aninteraction between a BAL molecule and a non-BAL molecule. Biologicallyactive portions of a BAL protein include peptides comprising amino acidsequences sufficiently homologous to or derived from the amino acidsequence of the BAL protein, e.g., the amino acid sequence shown in SEQID NO:2 or 5, which include less amino acids than the full length BALproteins, and exhibit at least one activity of a BAL protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the BAL protein, e.g., modulating cellular adhesion. Abiologically active portion of a BAL protein can be a polypeptide whichis, for example, 10, 25, 50, 100, 200 or more amino acids in length.Biologically active portions of a BAL protein can be used as targets fordeveloping agents which modulate a BAL mediated activity, e.g., theoccurrence or severity of a lymphoma, e.g., non-Hodgkin's lymphoma.

In one embodiment, a biologically active portion of a BAL proteincomprises at least one proline rich domain and/or at least one tyrosinephosphorylation site. It is to be understood that a preferredbiologically active portion of a BAL protein of the present inventionmay contain at least one of the above-identified structural domains. Amore preferred biologically active portion of a BAL protein may containat least two of the above-identified structural domains. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of a native BAL protein.

In a preferred embodiment, the BAL protein has an amino acid sequenceshown in SEQ ID NO:2 or 5. In other embodiments, the BAL protein issubstantially homologous to SEQ ID NO:2 or 5, and retains the functionalactivity of the protein of SEQ ID NO:2 or 5, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail in subsection I above. Accordingly, in another embodiment, theBAL protein is a protein which comprises an amino acid sequence at leastabout 50%, 55%, 60%, 62%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 98% or morehomologous to SEQ ID NO:2 or 5.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the BAL amino acidsequence of SEQ ID NO:2 or 5 having 177 amino acid residues, at least80, preferably at least 100, more preferably at least 120, even morepreferably at least 140, and even more preferably at least 150, 160 or170 amino acid residues are aligned). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to BAL nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to BAL proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The invention also provides BAL chimeric or fusion proteins. As usedherein, a BAL “chimeric protein” or “fusion protein” comprises a BALpolypeptide operatively linked to a non-BAL polypeptide. An “BALpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to BAL, whereas a “non-BAL polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the BAL protein, e.g., aprotein which is different from the BAL protein and which is derivedfrom the same or a different organism. Within a BAL fusion protein theBAL polypeptide can correspond to all or a portion of a BAL protein. Ina preferred embodiment, a BAL fusion protein comprises at least onebiologically active portion of a BAL protein. In another preferredembodiment, a BAL fusion protein comprises at least two biologicallyactive portions of a BAL protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the BAL polypeptideand the non-BAL polypeptide are fused in-frame to each other. Thenon-BAL polypeptide can be fused to the N-terminus or C-terminus of theBAL polypeptide.

For example, in one embodiment, the fusion protein is a GST-BAL fusionprotein in which the BAL sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant BAL.

In another embodiment, the fusion protein is a BAL protein containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of BAL can beincreased through use of a heterologous signal sequence.

The BAL fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. TheBAL fusion proteins can be used to affect the bioavailability of a BALsubstrate. Use of BAL fusion proteins may be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a BAL protein; (ii)mis-regulation of the BAL gene; and (iii) aberrant post-translationalmodification of a BAL protein.

Moreover, the BAL-fusion proteins of the invention can be used asimmunogens to produce anti-BAL antibodies in a subject, to purify BALligands and in screening assays to identify molecules which inhibit theinteraction of BAL with a BAL substrate.

Preferably, a BAL chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AnBAL-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the BAL protein.

The present invention also pertains to variants of the BAL proteinswhich function as either BAL agonists (mimetics) or as BAL antagonists.Variants of the BAL proteins can be generated by mutagenesis, e.g.,discrete point mutation or truncation of a BAL protein. An agonist ofthe BAL proteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a BAL protein.An antagonist of a BAL protein can inhibit one or more of the activitiesof the naturally occurring form of the BAL protein by, for example,competitively modulating a BAL-mediated activity of a BAL protein. Thus,specific biological effects can be elicited by treatment with a variantof limited function. In one embodiment, treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the BALprotein.

In one embodiment, variants of a BAL protein which function as eitherBAL agonists (mimetics) or as BAL antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a BAL protein for BAL protein agonist or antagonist activity. In oneembodiment, a variegated library of BAL variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of BAL variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential BAL sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of BAL sequences therein. There are avariety of methods which can be used to produce libraries of potentialBAL variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential BAL sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477.

In addition, libraries of fragments of a BAL protein coding sequence canbe used to generate a variegated population of BAL fragments forscreening and subsequent selection of variants of a BAL protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a BAL coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the BAL protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of BAL proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recrusive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify BAL variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated BAL library. For example, a library of expression vectors canbe transfected into a cell line which ordinarily responds to aparticular ligand in a BAL-dependent manner. The transfected cells arethen contacted with the ligand and the effect of expression of themutant on signaling by the ligand can be detected, e.g., by measuringcell survival or the activity of a BAL-regulated transcription factor.Plasmid DNA can then be recovered from the cells which score forinhibition, or alternatively, potentiation of signaling by the ligand,and the individual clones further characterized.

An isolated BAL protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind BAL using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length BAL protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of BAL for use as immunogens. Theantigenic peptide of BAL comprises at least 8 amino acid residues of theamino acid sequence shown in SEQ ID NO:2 or 5 and encompasses an epitopeof BAL such that an antibody raised against the peptide forms a specificimmune complex with BAL. Preferably, the antigenic peptide comprises atleast 10 amino acid residues, more preferably at least 15 amino acidresidues, even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofBAL that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity.

A BAL immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed BAL protein or a chemically synthesizedBAL polypeptide. The preparation can further include an adjuvant, suchas Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic BAL preparation induces a polyclonal anti-BAL antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-BALantibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as BAL. Examplesof immunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind BAL. The term “monoclonalantibody” or “monoclonal antibody composition”, as used herein, refersto a population of antibody molecules that contain only one species ofan antigen binding site capable of immunoreacting with a particularepitope of BAL. A monoclonal antibody composition thus typicallydisplays a single binding affinity for a particular BAL protein withwhich it immunoreacts.

Polyclonal anti-BAL antibodies can be prepared as described above byimmunizing a suitable subject with a BAL immunogen. The anti-BALantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized BAL. If desired, the antibody moleculesdirected against BAL can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-BAL antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem 0.255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique(Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a BAL immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds BAL.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-BAL monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature266:55052; Gefter et al. Somatic Cell Genet.., cited supra; Lerner, YaleJ. Biol. Med.., cited supra, Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindBAL, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-BAL antibody can be identified and isolated by screeninga recombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with BAL to thereby isolate immunoglobulinlibrary members that bind BAL. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol.Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram etal. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-BAL antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (I 987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (I 988) J. Immunol. 141:4053-4060.

An anti-BAL antibody (e.g., monoclonal antibody) can be used to isolateBAL by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-BAL antibody can facilitate thepurification of natural BAL from cells and of recombinantly produced BALexpressed in host cells. Moreover, an anti-BAL antibody can be used todetect BAL protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of the BALprotein. Anti-BAL antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling (i.e., physically linking) theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, -galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a BAL protein (ora portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cells and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., BAL proteins, mutantforms of BAL proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of BAL proteins in prokaryotic or eukaryotic cells. Forexample, BAL proteins can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:3140), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in BAL activity assays, (e.g.,direct assays or competitive assays described in detail below), or togenerate antibodies specific for BAL proteins, for example. In apreferred embodiment, a BAL fusion protein expressed in a retroviralexpression vector of the present invention can be utilized to infectbone marrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time as passed (e.g., six (6) weeks).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the BAL expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(InVitrogen Corp, San Diego, Calif.).

Alternatively, BAL proteins can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to BAL mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which a BALnucleic acid molecule of the invention is introduced, e.g., a BALnucleic acid molecule within a recombinant expression vector or a BALnucleic acid molecule containing sequences which allow it tohomologously recombine into a specific site of the host cell's genome.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aBAL protein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2nd, ed, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a BAL protein or can be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a BAL protein.Accordingly, the invention further provides methods for producing a BALprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding a BAL protein has beenintroduced) in a suitable medium such that a BAL protein is produced. Inanother embodiment, the method further comprises isolating a BAL proteinfrom the medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichBAL-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous BALsequences have been introduced into their genome or homologousrecombinant animals in which endogenous BAL sequences have been altered.Such animals are useful for studying the function and/or activity of aBAL and for identifying and/or evaluating modulators of BAL activity. Asused herein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, and the like. A transgene is exogenous DNA whichis integrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous BAL gene has been altered byhomologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing aBAL-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The BAL cDNAsequence of SEQ ID NO:1 or 4 can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a nonhuman homologue of ahuman BAL gene, such as a mouse or rat BAL gene, can be used as atransgene. Alternatively, a BAL gene homologue, can be isolated based onhybridization to the BAL cDNA sequences of SEQ ID NO:1, 3, 4, or 6(described further in subsection I above) and used as a transgene.Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to a BALtransgene to direct expression of a BAL protein to particular cells.Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a BAL transgene in its genome and/or expression of BAL mRNAin tissues or cells of the animals. A transgenic founder animal can thenbe used to breed additional animals carrying the transgene. Moreover,transgenic animals carrying a transgene encoding a BAL protein canfurther be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a BAL gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the BAL gene. The BAL gene can be a human gene(e.g., the cDNA of SEQ ID NO:3), but more preferably, is a non-humanhomologue of a human BAL gene (e.g., the cDNA of SEQ ID NO:6). Forexample, a mouse BAL gene can be used to construct a homologousrecombination nucleic acid molecule, e.g., a vector, suitable foraltering an endogenous BAL gene in the mouse genome. In a preferredembodiment, the homologous recombination nucleic acid molecule isdesigned such that, upon homologous recombination, the endogenous BALgene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thehomologous recombination nucleic acid molecule can be designed suchthat, upon homologous recombination, the endogenous BAL gene is mutatedor otherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous BAL protein). In the homologousrecombination nucleic acid molecule, the altered portion of the BAL geneis flanked at its 5′ and 3′ ends by additional nucleic acid sequence ofthe BAL gene to allow for homologous recombination to occur between theexogenous BAL gene carried by the homologous recombination nucleic acidmolecule and an endogenous BAL gene in a cell, e.g., an embryonic stemcell. The additional flanking BAL nucleic acid sequence is of sufficientlength for successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the homologous recombination nucleic acid molecule(see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The homologousrecombination nucleic acid molecule is introduced into a cell, e.g., anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced BAL gene has homologously recombined with the endogenousBAL gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). Theselected cells can then injected into a blastocyst of an animal (e.g., amouse) to form aggregation chimeras (see e.g., Bradley, A. inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA bygermline transmission of the transgene. Methods for constructinghomologous recombination nucleic acid molecules, e.g., vectors, orhomologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijistra et al.; and WO93/04169 by Berns et al.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The recontructed oocyte is then cultured such that it develops to morulaor blastocyte and then transferred to pseudopregnant female fosteranimal. The offspring borne of this female foster animal will be a cloneof the animal from which the cell, e.g., the somatic cell, is isolated.

IV. Pharmaceutical Compositions

The BAL nucleic acid molecules, fragments of BAL proteins, and anti-BALantibodies (also referred to herein as “active compounds”) of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a fragment of a BAL protein or an anti-BAL antibody) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (ie., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, a BAL protein of the invention has one or more of thefollowing activities: (1) it interacts with a non-BAL protein molecule,e.g., CRK, CRK-L, SHP-2, P13K, or ZAP70; (2) it activates aBAL-dependent signal transduction pathway; (3) it modulates theoccurrence and severity of a malignancy such as a lymphoma, e.g.,non-Hodgkin's lymphoma; and (4) it modulates cell migration, motility,and shape, as well as cell/cell and cell/extra-cellular matrixinteractions, and, thus, can be used to, for example, (1) modulate theinteraction with a non-BAL protein molecule; (2) activate aBAL-dependent signal transduction pathway; (3) modulate the occurrenceand severity of a malignancy such as a lymphoma, e.g., non-Hodgkin'slymphoma; and (4) modulate cell migration, motility, and shape, as wellas cell/cell and cell/extra-cellular matrix interactions.

The isolated nucleic acid molecules of the invention can be used, forexample, to express BAL protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect BAL mRNA(e.g., in a biological sample) or a genetic alteration in a BAL gene,and to modulate BAL activity, as described further below. The BALproteins can be used to treat disorders characterized by insufficient orexcessive production of a BAL substrate or production of BAL inhibitors.In addition, the BAL proteins can be used to screen for naturallyoccurring BAL substrates, to screen for drugs or compounds whichmodulate BAL activity, as well as to treat disorders characterized byinsufficient or excessive production of BAL protein or production of BALprotein forms which have decreased or aberrant activity compared to BALwild type protein (e.g., Non-Hodgkin's lymphoma). Moreover, the anti-BALantibodies of the invention can be used to detect and isolate BALproteins, regulate the bioavailability of BAL proteins, and modulate BALactivity.

A. Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying and/or producing modulators, i.e., candidate ortest compounds or agents (e.g., peptides, peptidomimetics, smallmolecules or other drugs) which bind to BAL proteins, have a stimulatoryor inhibitory effect on, for example, BAL expression or BAL activity, orhave a stimulatory or inhibitory effect on, for example, the expressionor activity of a BAL substrate.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a BAL protein or polypeptideor biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of a BAL protein or polypeptideor biologically active portion thereof. The test compounds of thepresent invention can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et at. (1993) Proc. Natl. Acad.Sci. USA. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a BAL protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate BAL activity is determined. Determining the ability of the testcompound to modulate BAL activity can be accomplished by monitoring, forexample, the survival of a cell which expresses BAL or the activity of aBAL-regulated transcription factor. The cell, for example, can be ofmammalian origin, e.g., a peripheral blood cell.

The ability of the test compound to modulate BAL binding to a substrateor to bind to BAL can also be determined. Determining the ability of thetest compound to modulate BAL binding to a substrate can beaccomplished, for example, by coupling the BAL substrate with aradioisotope or enzymatic label such that binding of the BAL substrateto BAL can be determined by detecting the labeled BAL substrate in acomplex. Determining the ability of the test compound to bind BAL can beaccomplished, for example, by coupling the compound with a radioisotopeor enzymatic label such that binding of the compound to BAL can bedetermined by detecting the labeled BAL compound in a complex. Forexample, compounds (e.g., BAL substrates) can be labeled with ¹²⁵I, ³⁵S,¹⁴C, or ³H, either directly or indirectly, and the radioisotope detectedby direct counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound (e.g., a BAL substrate) to interact with BAL without thelabeling of any of the interactants. For example, a microphysiometer canbe used to detect the interaction of a compound with BAL without thelabeling of either the compound or the BAL. McConnell, H. M. et al.(1992) Science 257:1906-1912. As used herein, a “microphysiometer”(e.g., Cytosensor) is an analytical instrument that measures the rate atwhich a cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a compound and BAL.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a BAL target molecule (e.g., a BALsubstrate) with a test compound and determining the ability of the testcompound to modulate (e.g. stimulate or inhibit) the activity of the BALtarget molecule. Determining the ability of the test compound tomodulate the activity of a BAL target molecule can be accomplished forexample, by determining the ability of the BAL protein to bind to orinteract with the BAL target molecule.

Determining the ability of the BAL protein or a biologically activefragment thereof, to bind to or interact with a BAL target molecule canbe accomplished by one of the methods described above for determiningdirect binding. In a preferred embodiment, determining the ability ofthe BAL protein to bind to or interact with a BAL target molecule can beaccomplished by determining the activity of the target molecule. Forexample, the activity of the target molecule can be determined bydetecting induction of a cellular second messenger of the target (ie.,intracellular Ca²⁺, diacylglycerol, IP₃, and the like), detectingcatalytic/enzymatic activity of the target an appropriate substrate,detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response.

In yet another embodiment, an assay of the present invention is acell-free assay in which a BAL protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to the BAL protein or biologically active portionthereof is determined. Preferred biologically active portions of the BALproteins to be used in assays of the present invention include fragmentswhich participate in interactions with non-BAL molecules, e.g.,fragments with high surface probability scores. Binding of the testcompound to the BAL protein can be determined either directly orindirectly as described above. In a preferred embodiment, the assayincludes contacting the BAL protein or biologically active portionthereof with a known compound which binds BAL to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a BAL protein, whereindetermining the ability of the test compound to interact with a BALprotein comprises determining the ability of the test compound topreferentially bind to BAL or biologically active portion thereof ascompared to the known compound.

In another embodiment, the assay is a cell-free assay in which a BALprotein or biologically active portion thereof is contacted with a testcompound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the BAL protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a BAL protein can beaccomplished, for example, by determining the ability of the BAL proteinto bind to a BAL target molecule by one of the methods described abovefor determining direct binding. Determining the ability of the BALprotein to bind to a BAL target molecule can also be accomplished usinga technology such as real-time Biomolecular Interaction Analysis (BIA).Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 andSzabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein,“BIA” is a technology for studying biospecific interactions in realtime, without labeling any of the interactants (e.g., BIAcore). Changesin the optical phenomenon of surface plasmon resonance (SPR) can be usedas an indication of real-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a BAL protein can be accomplishedby determining the ability of the BAL protein to further modulate theactivity of a downstream effector of a BAL target molecule. For example,the activity of the effector molecule on an appropriate target can bedetermined or the binding of the effector to an appropriate target canbe determined as previously described.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of isolated proteins (e.g., BALproteins or biologically active portions thereof). In the case ofcell-free assays in which a membrane-bound form of an isolated proteinis used it may be desirable to utilize a solubilizing agent such thatthe membrane-bound form of the isolated protein is maintained insolution. Examples of such solubilizing agents include non-ionicdetergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either BAL or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to a BAL protein, or interaction of aBAL protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/BAL fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or BAL protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of BALbinding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a BALprotein or a BAL target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated BAL protein ortarget molecules can be prepared from biotin-NHS(N-hydroxy-succinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with BAL protein or target molecules but which donot interfere with binding of the BAL protein to its target molecule canbe derivatized to the wells of the plate, and unbound target or BALprotein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the BAL protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the BAL protein or target molecule.

In another embodiment, modulators of BAL expression are produced oridentified in a method wherein a cell is contacted with a candidatecompound and the expression of BAL mRNA or protein in the cell isdetermined. The level of expression of BAL mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of BAL mRNA or protein in the absence of the candidatecompound. The candidate compound can then be produced or identified as amodulator of BAL expression based on this comparison. For example, whenexpression of BAL mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator of BALmRNA or protein expression (i.e. a stimulator of BAL mRNA or proteinexpression is produced). Alternatively, when expression of BAL mRNA orprotein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isproduced or identified as an inhibitor of BAL mRNA or protein expression(i.e. an inhibitor of BAL mRNA or protein expression is produced). Thelevel of BAL mRNA or protein expression in the cells can be determinedby methods described herein for detecting BAL mRNA or protein.

In yet another aspect of the invention, the BAL proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with BAL (“BAL-binding proteins” or “BAL-bp”) and are involvedin BAL activity. Such BAL-binding proteins are also likely to beinvolved in the propagation of signals by the BAL proteins or BALtargets as, for example, downstream elements of a BAL-mediated signalingpathway. Alternatively, such BAL-binding proteins are likely to be BALinhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a BAL protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a BAL-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the BALprotein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified and/or produced asdescribed herein in an appropriate animal model. For example, an agentidentified and/or produced as described herein (e.g., a BAL modulatingagent, an antisense BAL nucleic acid molecule, a BAL-specific antibody,or a BAL-binding partner) can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified and/or produced as described hereincan be used in an animal model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified and/or produced by the above-described screeningassays for treatments as described herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the BAL nucleotide sequences, described herein,can be used to map the location of the BAL genes on a chromosome(further described in Example 1, below). The mapping of the BALsequences to chromosomes is an important first step in correlating thesesequences with genes associated with disease.

Briefly, BAL genes can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp in length) from the BAL nucleotide sequences.Computer analysis of the BAL sequences can be used to predict primersthat do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers can then be usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the BAL sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the BALnucleotide sequences to design oligonucleotide primers, sublocalizationcan be achieved with panels of fragments from specific chromosomes.Other mapping strategies which can similarly be used to map a BALsequence to its chromosome include in situ hybridization (described inFan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the BAL gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The BAL sequences of the present invention can also be used to identifyindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the BAL nucleotide sequences described herein can be usedto prepare two PCR primers from the 5′ and 3′ ends of the sequences.These primers can then be used to amplify an individual's DNA andsubsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The BAL nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1 or 4can comfortably provide positive individual identification with a panelof perhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO:3 or 6 are used, a more appropriate number of primers forpositive individual identification would be 500-2,000.

If a panel of reagents from BAL nucleotide sequences described herein isused to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of Partial BAL Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 or 4 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include theBAL nucleotide sequences or portions thereof, e.g., fragments derivedfrom the noncoding regions of SEQ ID NO:1 or 4 having a length of atleast 20 bases, preferably at least 30 bases.

The BAL nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such BAL probes can be used to identify tissueby species and/or by organ type.

In a similar fashion, these reagents, e.g., BAL primers or probes can beused to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine:

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining BAL proteinand/or nucleic acid expression as well as BAL activity, in the contextof a biological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant BALexpression or activity, e.g., a malignancy such as a lymphoma, e.g.,non-Hodgkin's lymphoma. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping a disorder associated with BAL protein, nucleic acidexpression or activity. For example, mutations in a BAL gene can beassayed in a biological sample. Such assays can be used for prognosticor predictive purpose to thereby phophylactically treat an individualprior to the onset of a disorder characterized by or associated with BALprotein, nucleic acid expression or activity e.g., a malignancy such asa lymphoma e.g., non-Hodgkin's lymphoma.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of BAL inclinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of BAL proteinor nucleic acid in a biological sample involves obtaining a biologicalsample from a test subject and contacting the biological sample with acompound or an agent capable of detecting BAL protein or nucleic acid(e.g., mRNA or genomic DNA) that encodes BAL protein such that thepresence of BAL protein or nucleic acid is detected in the biologicalsample. A preferred agent for detecting BAL mRNA or genomic DNA is alabeled nucleic acid probe capable of hybridizing to BAL mRNA or genomicDNA. The nucleic acid probe can be, for example, a full-length BALnucleic acid, such as the nucleic acid of SEQ ID NO:1, 3, 4, or 6, or aportion thereof, such as an oligonucleotide of at least 15, 30, 50, 100,250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to BAL mRNA or genomic DNA. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein.

A preferred agent for detecting BAL protein is an antibody capable ofbinding to BAL protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect BAL mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of BAL mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of BAL protein includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of BAL genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of BAL protein includeintroducing into a subject a labeled anti-BAL antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting BAL protein, mRNA, orgenomic DNA, such that the presence of BAL protein, mRNA or genomic DNAis detected in the biological sample, and comparing the presence of BALprotein, mRNA or genomic DNA in the control sample with the presence ofBAL protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of BAL ina biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting BAL protein or mRNA in abiological sample; means for determining the amount of BAL in thesample; and means for comparing the amount of BAL in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectBAL protein or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant BAL expression or activity e.g., a malignancysuch as a lymphoma, e.g., non-Hodgkin's lymphoma. As used herein, theterm “aberrant” includes a BAL expression or activity which deviatesfrom the wild type BAL expression or activity. Aberrant expression oractivity includes increased or decreased expression or activity, as wellas expression or activity which does not follow the wild typedevelopmental pattern of expression or the subcellular pattern ofexpression. For example, aberrant BAL expression or activity is intendedto include the cases in which a mutation in the BAL gene causes the BALgene to be under-expressed or over-expressed and situations in whichsuch mutations result in a non-functional BAL protein or a protein whichdoes not function in a wild-type fashion, e.g., a protein which does notinteract with a BAL ligand or one which interacts with a non-BAL ligand.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in BALprotein activity or nucleic acid expression, e.g., a malignancy such asa lymphoma, e.g., non-Hodgkin's lymphoma. Alternatively, the prognosticassays can be utilized to identify a subject having or at risk fordeveloping a disorder associated with a misregulation in BAL proteinactivity or nucleic acid expression, such as a e.g., a malignancy suchas a lymphoma, e.g., non-Hodgkin's lymphoma. Thus, the present inventionprovides a method for identifying a disease or disorder associated withaberrant BAL expression or activity in which a test sample is obtainedfrom a subject and BAL protein or nucleic acid (e.g., mRNA or genomicDNA) is detected, wherein the presence of BAL protein or nucleic acid isdiagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant BAL expression or activity. As usedherein, a “test sample” refers to a biological sample obtained from asubject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant BAL expression or activity. For example, suchmethods can be used to determine whether a subject can be effectivelytreated with an agent for a e.g., a malignancy such as a lymphoma, e.g.,non-Hodgkin's lymphoma. Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant BAL expression or activity inwhich a test sample is obtained and BAL protein or nucleic acidexpression or activity is detected (e.g., wherein the abundance of BALprotein or nucleic acid expression or activity is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant BAL expression or activity).

The methods of the invention can also be used to detect geneticalterations in a BAL gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inBAL protein activity or nucleic acid expression, such as a e.g., amalignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a BAL-protein, or the mis-expression of the BAL gene.For example, such genetic alterations can be detected by ascertainingthe existence of at least one of 1) a deletion of one or morenucleotides from a BAL gene; 2) an addition of one or more nucleotidesto a BAL gene; 3) a substitution of one or more nucleotides of a BALgene, 4) a chromosomal rearrangement of a BAL gene; 5) an alteration inthe level of a messenger RNA transcript of a BAL gene, 6) aberrantmodification of a BAL gene, such as of the methylation pattern of thegenomic DNA, 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a BAL gene, 8) a non-wild type level of aBAL-protein, 9) allelic loss of a BAL gene, and 10) inappropriatepost-translational modification of a BAL-protein. As described herein,there are a large number of assays known in the art which can be usedfor detecting alterations in a BAL gene. A preferred biological sampleis a tissue or serum sample isolated by conventional means from asubject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the BAL-gene (seeAbravaya et al. (1995) Nucleic Acids Res .23:675-682). This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic DNA or mRNA) from the cells of thesample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a BAL gene under conditions such thathybridization and amplification of the BAL-gene (if present) occurs, anddetecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a BAL gene from a sample cellcan be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in BAL can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in BAL can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the BAL gene anddetect mutations by comparing the sequence of the sample BAL with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be used when performingthe diagnostic assays ((1995) Biotechniques 19:448), includingsequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol38:147-159).

Other methods for detecting mutations in the BAL gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type BAL sequence with potentially mutant RNA orDNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) MethodsEnzymol. 217:286-295. In a preferred embodiment, the control DNA or RNAcan be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in BAL cDNAs obtained from samplesof cells. For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cells cleaves Tat G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a BAL sequence,e.g., a wild-type BAL sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in BAL genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control BALnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method uses heteroduplex analysis to separatedouble stranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by the useof pre-packaged diagnostic kits which include at least one probe nucleicacid or antibody reagent described herein, which may be convenientlyused, e.g., in clinical settings to diagnose patients exhibitingsymptoms or family history of a disease or illness involving a BAL genesuch as non-Hodgkin's lymphoma. Such kits can optionally includeinstructions for use.

Furthermore, any cell type or tissue in which BAL is expressed may beused in the prognostic assays described herein.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a BAL protein can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent determined by a screening assay as described herein toincrease BAL gene expression, protein levels, or upregulate BALactivity, can be monitored in clinical trials of subjects exhibitingdecreased BAL gene expression, protein levels, or downregulated BALactivity. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease BAL gene expression, protein levels, ordownregulate BAL activity, can be monitored in clinical trials ofsubjects exhibiting increased BAL gene expression, protein levels, orupregulated BAL activity. In such clinical trials, the expression oractivity of a BAL gene, and preferably, other genes that have beenimplicated in, for example, a BAL-associated disorder can be used as a“read out” or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including BAL, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates BAL activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on BAL-associated disorders (e.g., malignanciessuch as non-Hodgkin's lymphoma), for example, in a clinical trial, cellscan be isolated and RNA prepared and analyzed for the levels ofexpression of BAL and other genes implicated in the BAL-associateddisorder, respectively. The levels of gene expression (e.g., a geneexpression pattern) can be quantified by northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods described herein, or bymeasuring the levels of activity of BAL or other genes. In this way, thegene expression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points duringtreatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a BAL protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the BAL protein, mRNA, or genomicDNA in the post-administration samples; (v) comparing the level ofexpression or activity of the BAL protein, mRNA, or genomic DNA in thepre-administration sample with the BAL protein, mRNA, or genomic DNA inthe post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increaseexpression or activity of BAL to lower levels than detected, i.e. todecrease the effectiveness of the agent. According to such anembodiment, BAL expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observablephenotypic response.

D. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant BAL expression or activitye.g., a malignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma.With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”.) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the BAL molecules ofthe present invention or BAL modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant BALexpression or activity, by administering to the subject a BAL moleculeor an agent which modulates BAL expression or at least one BAL activity.Subjects at risk for a disease which is caused or contributed to byaberrant BAL expression or activity can be identified by, for example,any or a combination of diagnostic or prognostic assays as describedherein. Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the BAL aberrancy, such thata disease or disorder is prevented or, alternatively, delayed in itsprogression. Depending on the type of BAL aberrancy, for example, a BALmolecule, BAL agonist, or BAL antagonist can be used to treat thesubject. The appropriate agent can be determined based on, for example,the screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating BALexpression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a BAL or agent that modulates one or more of theactivities of BAL protein activity associated with the cell. An agentthat modulates BAL protein activity can be an agent as described herein,such as a nucleic acid or a protein, a naturally-occurring targetmolecule of a BAL protein (e.g., a BAL substrate), a BAL antibody, a BALagonist or antagonist, a peptidomimetic of a BAL agonist or antagonist,or other small molecule. In one embodiment, the agent stimulates one ormore BAL activities. Examples of such stimulatory agents include activeBAL protein and a nucleic acid molecule encoding BAL that has beenintroduced into the cell. In another embodiment, the agent inhibits oneor more BAL activities. Examples of such inhibitory agents includeantisense BAL nucleic acid molecules, anti-BAL antibodies, and BALinhibitors. These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of a BALprotein or nucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulate (e.g.,upregulate or downregulate) BAL expression or activity. In anotherembodiment, the method involves administering a BAL protein or nucleicacid molecule as therapy to compensate for reduced or aberrant BALexpression or activity.

Stimulation of BAL activity is desirable in situations in which BAL isabnormally downregulated and/or in which increased BAL activity islikely to have a beneficial effect. For example, stimulation of BALactivity is desirable in situations in which a BAL molecule isdownregulated and/or in which increased BAL activity is likely to have abeneficial effect. Likewise, inhibition of BAL activity is desirable insituations in which BAL is abnormally upregulated and/or in whichdecreased BAL activity is likely to have a beneficial effect.

3. Pharmacogenomics

The BAL molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on BAL activity(e.g., BAL gene expression) as identified by a screening assay describedherein can be administered to individuals to treat (prophylactically ortherapeutically) BAL-associated disorders (e.g., e.g., malignancies suchas non-Hodgkin's lymphoma). In conjunction with such treatment,pharmacogenomics (i e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a BAL molecule or BALmodulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a BAL molecule or BAL modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a BALprotein of the present invention), all common variants of that gene canbe fairly easily identified in the population and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a BAL molecule orBAL modulator of the present invention) can give an indication whethergene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a BAL molecule orBAL modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing areincorporated herein by reference.

EXAMPLES

The following Materials and Methods were used in the Examples Describedherein.

Cell lines and Primary Tumor Specimens

Human DLB-CL cell lines DHL-4, DHL-7, DHL-8, DHL-10, HT, and theBurkitt's cell line Namalwa, were cultured in RPM1 1640 supplementedwith 10% heat-inactivated fetal calf serum, 2 mM glutamine, 1 mM sodiumpyruvate, 10 mM Herpes buffer and penicillin/streptomycin. Cryopreservedprimary tumor specimens were obtained from DLB-CL patients with knownclinical prognostic characteristics and long-term follow-up. Total RNAwas isolated from the cell lines and primary tumors as described inAguiar (1999) Blood 94(7):2403-13.

Differential Display, cDNA Cloning and Sequencing

Differential display was performed as described in Liang (1992) Science257(5072):967-71, and Aguiar, Blood (supra). The relevant differentialdisplay product was used as a probe to screen a size-selected anti-Igactivated normal B-cell cDNA library. BAL full length cloning wascompleted with 5′ and 3′ RACE PCR, performed as described in Aguiar,Blood (supra). All DNA sequencing was performed and analyzed on anApplied Biosystems model 373A automated sequencer (Perkin-ElmerCorporation, Norwalk, Conn.).

Northern Blot Analysis

Total RNA from DLB-CL cell lines and primary tumors was isolated,size-fractionated in 1% Agarose/formaldehyde gels and transferred tonylon membranes as described in Aguiar, Blood (supra). These membranesand additional multiple tissue northern blots (Clontech, Palo Alto,Calif.) were hybridized according to standard protocols with either adifferential display fragment probe, an 800 bp BAL probe (nucleotides900 to 1700) or B-actin.

PAC Library Screening, FISH and Somatic Cell Hybrid Mapping

The human PAC library RPCI1 (UK HGMP Resource Centre, Cambridge UK) wasscreened with a BAL cDNA probe according to standard protocols. DNA frompositive clones was Southern blotted and re-probed with a distinct BALprobe to confirm the specificity of the clones. PAC DNAs were Biotinlabeled, and hybridization of human normal metaphases performed asdescribed in Fletcher (1998) Nature Genetics 18(1):84-7. Image analysiswas performed with a cooled CCD camera (Photometrics) in conjunctionwith an image analysis system (Oncor). A human monochromosomal somaticcell hybrid DNA panel (UK HGMP Resource Centre, Cambridge UK) wasscreened by PCR according to the manufacturer's instructions.

Semi-quantitative Duplex RT-PCR

cDNAs from DLB-CLs patients with known clinical prognosticcharacteristics and long-term follow-up and from DLB-CL cell lines weresynthesized as described in Aguiar (1996) British J. Haematol.95(4):673-7. To control for the quantity and quality of input cDNA andthe amplification efficiency in individual test tubes, BAL cDNA wasco-amplified with the constitutively expressed ABL gene. Duplex RT-PCRproducts were electrophoresed in 2% agarose gels, blotted and hybridizedto internal BAL and ABL oligonucleotide probes. The abundance of BAL ina given sample was determined by comparing the intensity of theco-amplified PCR products with scanning densitometry. The sensitivity ofthe duplex RT-PCR was determined by constructing and analyzing astandard dilutional curve. In brief, fixed amounts of BAL negative andBAL positive cell line cDNAs were added to mimic BAL losses of 10%-100%.Upon co-amplification, the ratio of the intensity of the two bandsplotted against the percentage of BAL “loss” yields a straight line andr2 value of 0.967, indicating the power of this system in detectingreduced BAL expression in the patient samples.

Transfections, Western Blot and Fluorescence Microscopy

Full length BAL cDNA was cloned into the green fluorescent protein (GFP)expression vector pEGFP-C (Clontech, Polo Alto, Calif.) and into theuntagged expression vector pRc/CMV (Invitrogen, Carlsbad, Calif.).Linearized DNA from BAL-GFP and BAL-pRc/CMV constructs were transfectedby electroporation into the Namalwa B-cell lymphoma line and selectedwith G418 (Sigma, St Louis, Mo.). Thereafter, the stable BAL-GFP orGFP-only bulk transfectants were sorted to select high GFP expressingcells. The pRcCMV transfectants were cloned by limiting dilution asdescribed in Aguiar, Blood (supra). Total cell lysates, membrane,cytoplasm, and nuclear fractions from multiple BAL-GFP or GFP-onlytransfectants were obtained and western blots performed as described inAguiar, Blood (supra). Rabbit polyclonal anti-GFP anti-sera (Clontech)was used for immunological detection of BAL fusion proteins. The mousefibroblast NIH3T3 cells were seeded on glass coverslides and transfectedwith BAL-GFP or GFP-alone constructs by using lipofectin (Gibco).Forty-eight hours after transfection, the slides were rinsed withice-cold phosphate-buffered saline (PBS)-0.1% NaN₃, cells were fixedwith 3% paraformaldehyde and analyzed by fluorescence microscopy.

Cell Migration Assays

Multiple BAL expressing stable transfectants (BAL-GFP and BAL intopRc-CMV, selected on the basis of protein expression) and vector-onlycontrols (pEGFP and pRcCMV, respectively) were seeded overnight at 1×10⁶cells/ml in RPMI 10% FBS. Then, 2×10⁶/ml cells were starved inserum-free AIM-V medium (Gibco BRL, Gaithersburg, Md.), for 1 hour at37° C. in 5% CO₂. Migration assays were performed using 8 μ pore filters(6.5 mm Transwell, polycarbonate membrane, Costar, Cambridge Mass.).Cell suspensions (2×10⁵ in 100 μL) were plated into the upper chamber,whereas 600 μL of medium with or without recombinant human SDFI-α (100ng/mL) (R & D Systems, Minneapolis, Minn.) was added to the lowercompartment. The transwells were incubated for 4 hours at 37° C. in 5%CO₂. Thereafter, cells in the lower chamber were recovered and counted(Coulter automatic cell and particle counter). All clones (with andwithout SDFI-α) were analyzed in duplicate and the entire assay repeatedthree times.

Example 1 Identification and Characterization of BAL cDNA

In this example, the identification and characterization of the genesencoding human and murine BAL is described. To identify genes whichcontribute to the observed differences in clinical outcome in DLB-CLs,the technique of differential display (Liang P. et al. (1992) Science,257:967) was used in panels of primary tumors from patients with knownclinical prognostic characteristics and mature follow-up. BAL was foundto be significantly more abundant in tumors from patients with“high-risk (HR)” (International Prognostic Index, IPI) fatal diseasethan in tumors from cured “low risk (LR [IPI])” patients (Shipp M. etal. (1993) N. Engl. J. Med., 329:987-994).

In confirmatory northern analyses, primary tumors from cured “LR”patients consistently expressed low levels of BAL whereas tumors from“HR” patients with fatal disease consistently expressed high levels ofBAL (see FIG. 5). However, only 1 of 5 DLB-CL cell lines (DHL-7)expressed high levels of BAL. This observation was of particularinterest because DHL-7 grows as a semi-adherent monolayer whereasBAL-negative DLB-CL cell lines grow in suspension. These findingssuggest that BAL can be upregulated when DLB-CL cells interact withother cellular or extracellular components in vivo. Consistent with thishypothesis, tumors derived from a DLB-CL cell line grown in SCID miceexpress significantly higher levels of BAL than the parental suspensioncells (see FIG. 6).

In addition to being differentially expressed in high-risk and low-riskprimary DLB-CLs, BAL was expressed at higher levels in normalanti-Ig-activated splenic B-cells than in non-activated splenic B-cells(see FIG. 5). For this reason, the full length BAL cDNA was cloned byprobing an Ig-activated B-cell cDNA library with the 3′ BAL differentialdisplay product. Several overlapping cDNA clones were identified and 5′RACE PCR (described in, for example, Ishimaru F. et al. (1995) Blood85:3199-3207) was used to complete the full length BAL cDNA sequence.Two alternatively spliced BAL cDNAs of 3243 bp (BAL_(L)) and 3138 bp(BAL_(S)) were identified. These cDNAs encode previously uncharacterized854 aa and 819 aa proteins. In vitro translation experiments confirmedthat BAL cDNAs encode ˜85-87 kd proteins.

For the Bal human cDNA, two human cDNA libraries derived fromanti-immunoglobulin activated splenocytes and the Raji Burkitts lymphomacell line cloned into pCDM8 were screened to obtain additional fulllength Bal cDNAs.

For the Bal murine cDNAs, the BAL human sequence was used to search themouse EST database. A 418 bp clone (Accession Number AA475710, Soaresmouse mammary gland) homologous to the human sequence was identified.This sequence was used as “anchor” to several rounds of 5′ and 3′ RACEassays performed with mouse (Balb-c) spleen cDNA. (The sequencesobtained from the EST database were used as primers).

The nucleotide sequence encoding the human BAL protein is shown in FIG.1 and is set forth as SEQ ID NO:1. The full length protein encoded bythis nucleic acid comprises about 866 amino acids and has the amino acidsequence shown in FIG. 1 and set forth as SEQ ID NO:2. The coding region(open reading frame) of SEQ ID NO:1 is set forth as SEQ ID NO:3.

The nucleotide sequence encoding the mouse BAL protein is shown in FIG.2 and is set forth as SEQ ID NO:4. The full length protein encoded bythis nucleic acid comprises about 866 amino acids and has the amino acidsequence shown in FIG. 2 and set forth as SEQ ID NO:5. The coding region(open reading frame) of SEQ ID NO:4 is set forth as SEQ ID NO:6.

Tissue Distribution of BAL

Although BAL transcripts were detected in the majority of organs on amultiple tissue northern blot, BAL transcripts were most abundant inlymphoid organs (spleen, lymph colon mucosa, node, fetal liver, andperipheral blood) and several additional non-hematopoietic organs(heart, skeletal muscle) (see FIG. 8).

Subcellular Localization of BAL

To determine the subcellular location of the BAL protein, the longer andthe shorter BAL cDNAs (BAL_(L) and BAL_(S)) were cloned into the greenfluorescent protein (GFP) expression vector (pEGFP, Clontech) andtransiently transfected in NIH3T3 fibroblasts. These fibroblasts werethen examined by fluorescence microscopy. Fibroblasts were chosen forBAL subcellular localization because the lymphoma cell lines have scantcytoplasm, precluding optimal microscopic detection of subcellularstructures. BAL_(S) was found to localize to the nucleus (confirmed withwestern blotting of cellular subfractions) whereas BALL localized inboth the nucleus and the perinuclear cytoplasm. These data indicate thatin NIH 3T3 cells, BAL does not interact directly with the cytoskeletalnetwork, demonstrating that BAL may either influence cellular migrationin an indirect manner, or may traffic between the cytoplasm and thenucleus and directly modulate cell migration via cytoskeletoninteractions following specific signals.

BAL Promotes Homotypic Aggregation

In additional experiments, aggressive lymphoma cell lines weretransfected with pRc-CMV-BAL_(L) constructs and evaluated for changes inmorphology. pRc-CMV-BAL_(L) transfectants exhibited markedly increasedhomotypic aggregation, indicating that BAL enhances the adhesion ofthese cells. These observations are of particular interest because BALwas upregulated in DLB-CL tumors in SCID mice (see FIG. 6) and wassignificantly more abundant in primary DLB-CLs and the adherent DLB-CLcell line than in DLB-CL suspension cell lines (see FIG. 5).

Mapping of the BAL Locus

To map the BAL locus, a BAL cDNA probe was used to screen a humangenomic DNA PAC library (RPCI1). Genomic BAL-positive PAC clones wereused to perform FISH (fluorescence in situ hybridization) on normalhuman metaphases. In complementary experiments, a somatic cell hybridpanel was analyzed for BAL sequences. The BAL locus was mapped tochromosome 3q21 (see FIG. 9), an area of known abnormalities in multiplehematologic malignancies, including DLB-CL and other aggressive B-celllymphomas (Cabanillas F et al. (1988) Cancer Res., 48:5557-5564;Schouten H. et al. (1990) Blood, 75:1841; Monni O. et al. (1998), Genes,Chrom. & Cancer, 21:298-307; and (Mitelman F. et al. (1997) Nat. Genet,15:417-474).

Analysis of the Human BAL Molecules

A BLASTP 2.0.6 search using an e-value threshold for inclusion of 0.001and a word length of 854 letters (Altschul et al. (1990) J. Mol. Biol.215:403) of the protein sequence of human BAL revealed that human BAL issimilar to the histone macro-H2A.1 protein (Accession Number Q02874).The human BAL protein is 26% identical to the histone macro-H2A.1protein (Accession Number Q02874) over amino acid residues 319-485 and22% identical over amino acid residues 160-288.

A BLASTN 2.0.5 search using an e-value threshold for inclusion of 9e-⁰⁹,score 61) and a word length of 3244 letters (Altschul et al. (1990) J.Mol. Biol. 215:403) of the nucleotide sequence of human BAL revealedthat BAL is similar to the Soares pregnant uterus NbHPU Homo sapienscDNA clone 502921 (Accession Number AA 151346). The human BAL nucleicacid molecule is 98% identical to the Soares pregnant uterus NbHPU Homosapiens cDNA clone 502921 (Accession Number AA151346) over nucleotides2528 to 3132.

A BLASTP 2.0.6 search using an e-value threshold for inclusion of 0.001and a word length of 866 letters (Altschul et al. (1990) J. Mol. Biol.215:403) of the protein sequence of murine BAL revealed that the murineBAL is similar to the histone macro-H2A. 1 protein (Accession NumberQ02874). The murine BAL protein is 26% identical to the histonemacro-H2A.1 protein (Accession Number Q02874) over amino acid residues261-450 and 24% identical over amino acid residues 74-250.

A BLASTN 2.0.5 search using an e-value threshold for inclusion of 1e⁻⁰⁸,score 60) (Altschul et al. (1990) J. Mol. Biol. 215:403) of thenucleotide sequence of murine BAL revealed that BAL is similar to theSoares 2NbMT Mus musculus cDNA clone 1446050 (Accession Number A157103). The murine BAL nucleic acid molecule is 99% identical to theSoares 2NbMT Mus musculus cDNA clone 1446050 (Accession Number AI157103)over nucleotides 2295 to 2739.

The human BAL protein was aligned with the murine BAL protein using theALIGN program (version 2.0), a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 2. The results showed a 61.5%identity between the two sequences (see FIG. 3).

The human BAL nucleic acid molecule was aligned with the murine BALnucleic acid molecule using the ALIGN program (version 2.0), a PAM120weight residue table, a gap length penalty of 16 and a gap penalty of 4.The results showed a 71.7% identity between the two sequences (see FIG.4).

Analysis of the predicted 819 aa human BAL protein indicates thatspecific regions of human BAL have partial homology to two previouslycharacterized domains in otherwise related proteins (FIG. 7). The humanBAL N-terminal region (aa 136-256 and 335-447) contains a duplicateddomain of unknown function (Pfam at www.wustl.edu) which is found in thenon-histone region of histone Macro H2A, non-structural polyproteins ofssRNA viruses or on its own in a family of proteins from bacteria toeukaryotes (Pehrson J. R. et al. (1999) Nucleic Acids Research26:2837-2842). Taken together, these data indicate that thisevolutionarily conserved, as yet uncharacterized, domain is involved inan important and ubiquitous cellular process.

The human BAL C-terminal region (aa 508-709), is partially homologous toprotein families (myosin heavy chain and cytoskeleton linkersezrin-radixin-moesin [ERM]) (see FIG. 7) which primarily governphysically integrated cellular functions such as cell migration,motility, and shape, as well as cell/cell and cell/extra-cellular matrixinteractions through adhesion molecules. Specifically, BAL is homologousto the rod domain (filament forming properties) of alpha and betacardiac myosin (Warrick et al. (1987) Annual Rev. Cell Biol. 3:379-421)and to the alpha-helical region of moesin, the most abundant ERM proteinin lymphocytes (Bretscher (1999) Current Biology 8(12):721-4).

Example 2 Identification of Co-associating Molecules and SignalingPathways Relevant to BAL Biological Activity

The following experiments are designed to confirm that: (1) BAL isphosphorylated on tyrosine; (2) BAL co-associates with other tyrosinephosphoproteins; (3) BAL specifically co-associates with CRK-L, SHP-2,P13K or ZAP70; and (4) BAL is a major component of signaling pathways inlymphohematopietic cells.

Generation of BAL_(HA) and BAL_(GRP) DLB-CL Transfectants

BAL_(S) and BAL_(L) cDNAs are cloned into the GFP- and HA-taggedexpression vectors (pEGFP, Clontech and pHM6, Boehringer) andtransfected into pEGFP-BAL and MH6-BAL into DLB-CL cell lines thatexpress the protein (DHL-7) or lack endogenous BAL expression (DHL-4).Both BAL_(GFP) and BAL_(HA) transfectants are evaluated in order toconfirm BAL nuclear and perinuclear cytoplasmic localization in DLB-CLs.To characterize BAL function, BAL_(HA) is preferentially used becauseits smaller tagged protein is more likely to retain the physiologicallyrelevant binding sites.

Tyrosine Phosphorylation of BAL and Additional Co-associated Proteins

To confirm that BAL itself is tyrosine phosphorylated and that BALco-associates with additional tyrosine phosphoproteins, BAL isimmunoprecipitated from untreated or anti-Ig-treated DHL-7 and DHL-4pHM6-BAL transfectants using an HA monoclonal antibody. Thereafter, theBAL_(HA) immunoprecipitates are immunoblotted with a phosphotyrosineantibody, 4G10 (Upstate Biotechnology, Inc.). The molecular weights ofidentified tyrosine phosphoproteins are compared to that of BAL andadditional candidate co-associated tyrosine phosphoproteins. Incomplementary experiments, BAL_(HA) immunoprecipitates are blotted withspecific antibodies to potential co-associating tyrosine phosphoproteinssuch as CRK/CRK-L, SHP-2, P13K, ZAP70 and additional candidates withmolecular weights that are similar to those of identified BALco-associated phosphoproteins. Because it may be easier to isolateco-associated proteins with concentrated highly purified recombinantBAL, the BAL_(GST) fusion protein beads (described above) are alsoincubated with DHL-7 cell lysates, the BAL complexes are blotted, andresulting blots are probed with a phosphotyrosine antibody (4G10).

Candidate co-associating proteins can further be identified using theyeast two-hybrid system (Matchmaker Two-Hybrid System by Clontech) asdescribed in Frederickson, R. (1998) Curr. Opin. Biotechnol., 9:90-96).

Tumorigenicity of BAL Transfectants

In addition to evaluating the role of BAL in specific signalingcascades, BAL's potential effects on the local growth and distantmetastasis of DLB-CL cell lines are further determined in an in vivomurine model. The above-mentioned pRc-CMV brief, parental, vector-only,pRc-CMV-BAL_(S) and BAL_(L) DLB-CL transfectants are injectedsubcutaneously or via tail vein into cohorts of SCID mice. Antisense BALconstructs can also be used because, as described herein, endogenous BALis upregulated when the suspension DLB-CL cell lines form local tumorsin vivo (see FIG. 6). Local tumorigenicity and distant metastasis can bescored at periodic intervals as described in Yakushijin Y. et al. (1998)Blood, 91:4282-4291.

Example 3 Analysis of BAL Expression at RNA and Protein Levels in anExpanded Series of Aggressive NHLs from well Characterized UniformlyTreated Patients

Semi-Quantitative Duplex RT-PCR

A semi-quantitative duplex RT-PCR assay (as described in Aguiar, R. etal. (1997) Blood 90 (Suppl. 1):491a and Aguiar, R. et al. (1997)Leukemia, 11:233-238) was performed to access BAL expression in a largeseries of primary DLB-CLs. Briefly, the “target sequence” (BAL) wasco-amplified with the constitutively expressed “control” ABL gene. ThePCR products were size-fractionated, blotted and hybridized withinternal BAL and ABL oligonucleotides. Autoradiogram signals werecaptured using a CCD camera linked to a frame grabber and intensitieswere quantified using the program NIH Image 1.55 (NIH, Bethesda, Md.)(as described in Aguiar, R. et al. (1997) Leukemia, 11:233-238). Toestablish the sensitivity of the assay, a series of dilutional controlswere constructed by mixing fixed amounts of cDNA from the BAL-negativeand BAL-positive DLB-CL cell lines. These controls mimic BAL losses ateach 10% interval up to 100% (see FIG. 10). When the ratio of BAL/ABLsignal was plotted against the percentage of BAL present in each ofthese test samples, it yielded a straight line and r² value of 0.967,confirming the sensitivity of the assay (see FIG. 10). The abundance ofBAL in a given sample is determined by comparing the ratio of intensityof co-amplified BAL and ABL signals and correlating this ratio with thatin the dilutional controls (as described in Aguiar, R. et a. (1997)Blood 90 (Suppl. 1):491a and Aguiar, R. et al. (1997) Leukemia,11:233-238).

To extend the initial observation regarding the risk related expressionof BAL in DLBCLs, a larger series of 28 additional primary DLB-CL withwell-characterized risk profiles and long term followup were studied(see FIG. 11). In these tumors, BAL expression, as determined by theratio of the intensity of the two co-amplified cDNAs (quantified withscanning densitometry) correlated closely with the clinical risk profile(see FIG. 12). BAL transcripts were significantly more abundant in highintermediate/high risk primary DLB-CLs than in cured low and lowintermediate risk tumors (p=0.0023, FIG. 12).

Development of a BAL Antibody

In order to generate a BAL antibody, the BAL cDNA was cloned into thepGEX expression vector (GST Gene Fusion System, Pharmacia). Aftersequencing the construct to ensure that the fusion was in frame and thatno mutations had been introduced in the BAL sequence, bacterial culturescontaining pGEX-BAL were treated with IPTG(isopropyl-1-Thio-b-D-Galactopyearanoside) and the GST-Bal fusionprotein was induced and affinity-purified on gluthathione-S-agarosebeads. Balb-c mice are immunized with the affinity-purified GST-BALprotein and their spleens harvested for generation of BAL monoclonalantibodies. Monoclonal antibodies are initially screened for reactivitywith pGEX-BAL and not with pGEX alone using ELISA. Positive hybridomasupernatants are then screened against immunoblotted parental and BALDLB-CL transfectants for reactivity with the appropriate-sized BALprotein.

Immunoperixidase Staining of Primary DLB-CLs for BAL

Immunoperoxidase staining of primary tumor specimens from a large seriesof well-characterized DLB-CL patients, can also be performed using theBAL monoclonal antibody produced as described above.

Example 4 Evaluation of the Integrity of the BAL Locus in AggressiveNHLs with 3q21 Abnormalities

BAL maps to chromosome band 3 q21, a region which is frequentlyassociated with non-random abnormalities (gain, loss and translocations)in NHLs (Cabanillas F et al. (1988) Cancer Res., 48:5557-5564);(Schouten H. et al (1990) Blood, 75:1841); (Monni O. et al. (1998),Genes, Chrom. & Cancer, 21:298-307); and (Mitelman F. et al. (1997) Nat.Genet, 15:417-474). To determine whether BAL is the target of thedescribed 3q21 abnormalities in aggressive NHLs, the integrity of theBAL locus is assessed in informative samples. The patient's metaphasesare initially probed with PAC clones encompassing the BAL locus usingthe FISH technique (Aguiar, R. et al. (1997) Blood, 90:3130-3135;Carapeti, M. et al. (1998) Blood, 91:3127-3133; Aguiar, R. et al (1997)Genes, Chrom. & Cancer, 20:408-411). In the case a translocationinvolving the BAL locus is detected, Southern blotting is used to mapthe breakpoint in BAL. Thereafter, strategies to clone the translocationpartner are used including RACE PCR and, if necessary, construction andscreening of patient tumor cDNA libraries with BAL probes (Aguiar, R. etal. (1997) Blood, 90:3130-3135 and Carapeti, M. et al. (1998) Blood,91:3127-3133. If loss or gain of DNA material is found with the FISH,Southern blots are performed to confirm these findings and DNA markersflanking the BAL locus are used to delineate the smallest region of lossor gain in the tumor specimens. To define the size of the region of gainor loss of 3q21 in informative samples, candidate 3q21 YAC clones areidentified from the Human Genomic Mapping Project databases(www.genome.wi.mit.edu, www.genethon.fr). Southern blot and PCR analysisof these clones' DNA maps BAL to a particular YAC (Aguiar, R. et al.(1997) Blood 90 (Suppl. 1):491 a). Thereafter, STS markers alreadyanchored to that particular YAC are identified in the relevant databases(www.genome.wi.mit.edu) and used as probes or PCR targets to determinetheir copy number in informative primary tumors as previously described(Aguiar, R. et al. (1997) Leukemia, 11:233-238). Using this strategy,the smallest region of loss or gain at 3q21 is defined, and whether BALis the target for these structural abnormalities is determined.

Example 5 Role of BAL in Modulating Cellular Motility and Migration

To investigate the potential role of BAL in modulating cellular motilityand migration, BAL_(S) was cloned into a GFP vector (pEGFP) and anuntagged expression vector (pRc-CMV) and pEGFP-BAL_(S), pRc-CMV BAL, andvector only transfectants were generated in an aggressive lymphoma cellline that constitutively expresses low levels of BAL (see FIG. 13). Theeffects of BAL overexpression on the migration of these transfectantswas investigated using a transwell system. In initial experiments,BAL_(S)-GFP or GFP-only transfectants were plated in the upper chamberand analyzed for migration to the lower chamber in the presence of thehematopoietic chemoattractant factor, stromal derived factor 1-α(SDF-1α) (see FIG. 14). In multiple independent assays, aggressivelymphoma BAL_(S)-GFP transfectants migrated at a 2-4 times higher ratethan the GFP-alone transfectants (p<0.01) (see FIG. 14). Similar resultswere obtained with multiple BAL_(S)pRcCMV transfectants indicating thatthe GFP moiety did not affect BAL function or the observed results.Taken together, these data indicate that BAL overexpression increasesthe migration of an aggressive NHL cell line.

Example 6 Expression of Recombinant BAL Protein in Bacterial Cells

In this example, BAL is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, BAL isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-BAL fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 7 Expression of Recombinant BAL Protein in COS Cells

To express the BAL gene in COS cells, the pcDNA/Amp vector by InvitrogenCorporation (San Diego, Calif.) is used. This vector contains an SV40origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire BAL protein and an HA tag (Wilson et al. (1984) Cell 37:767) or aFLAG tag fused in-frame to its 3′ end of the fragment is cloned into thepolylinker region of the vector, thereby placing the expression of therecombinant protein under the control of the CMV promoter.

To construct the plasmid, the BAL DNA sequence is amplified by PCR usingtwo primers. The 5′ primer contains the restriction site of interestfollowed by approximately twenty nucleotides of the BAL coding sequencestarting from the initiation codon; the 3′ end sequence containscomplementary sequences to the other restriction site of interest, atranslation stop codon, the HA tag or FLAG tag and the last 20nucleotides of the BAL coding sequence. The PCR amplified fragment andthe pcDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the BAL gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5a, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the BAL-pcDNA/Amp plasmidDNA using the calcium phosphate or calcium chloride co-precipitationmethods, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Other suitable methods for transfecting host cells canbe found in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Theexpression of the BAL polypeptide is detected by radiolabelling(³⁵S-methionine or ³⁵S-cysteine available from NEN, Boston, Mass., canbe used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly,the cells are labelled for 8 hours with ³⁵S-methionine (or³⁵S-cysteine). The culture media are then collected and the cells arelysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS,0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the BAL coding sequence is cloned directlyinto the polylinker of the pCDNA/Amp vector using the appropriaterestriction sites. The resulting plasmid is transfected into COS cellsin the manner described above, and the expression of the BAL polypeptideis detected by radiolabelling and immunoprecipitation using a BALspecific monoclonal antibody.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated nucleic acid molecule comprising the nucleotide sequenceset forth in SEQ ID NO: 1 or a full-length complement thereof.
 2. Anisolated nucleic acid molecule which encodes a polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 2, or a full-lengthcomplement thereof.
 3. An isolated nucleic acid molecule comprising thenucleotide sequence set forth in SEQ ID NO:3 or a full-length complementthereof.
 4. An isolated nucleic acid molecule consisting of thenucleotide sequence set forth in SEQ ID NO:1 or a full-length complementthereof.
 5. An isolated nucleic acid molecule consisting of thenucleotide sequence set forth in SEQ ID NO:3 or a full-length complementthereof.
 6. An isolated nucleic acid molecule which encodes apolypeptide consisting of the amino acid sequence set forth in SEQ IDNO: 2, or a full-length complement thereof.